Umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, nucleotide sequence, application, medicine and pharmaceutical composition

Abstract

The present invention discloses an umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, a nucleotide sequence, an application, a medicine, and a pharmaceutical composition. The amino acid sequence of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 is shown in SEQ ID No: 1. The peptide is an endogenous polypeptide derived from dynein heavy chain 5 and has minimal toxic and side effects. The peptide can be used to prepare a medicine and a corresponding pharmaceutical composition for restoring and maintaining vascular endothelial integrity and function or alleviating preeclampsia, and can effectively improve the angiogenesis ability of damaged endothelial cells and reduce the expression of vascular endothelial injury markers.

Description

Technical Field

[0001] The present invention relates to endogenous polypeptides, and in particular to an umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, a nucleotide sequence, an application, a medicine and a pharmaceutical composition. Background Art

[0002] Preeclampsia (PE) is a common and endemic condition during pregnancy, clinically manifested by hypertension and proteinuria. In severe cases, it can lead to maternal damage to vital organs (such as stroke, liver and kidney damage, and heart failure), as well as placental abruption, fetal growth restriction, and premature birth. It causes approximately 76,000 maternal and 500,000 perinatal deaths annually, posing a serious threat to maternal and fetal safety. Furthermore, preeclampsia significantly increases the mother's risk of long-term cardiovascular disease, type 2 diabetes, dyslipidemia, and premature death. Its offspring also experience a significantly increased risk of cardiovascular disease, diabetes, and obesity in adulthood, with a six-fold increased mortality risk, significantly impacting both the quality of life and the quality of life of the newborn. Despite the significant harms of preeclampsia, its pathogenesis remains incompletely understood, and existing clinical treatments are limited to symptomatic management, failing to effectively curb the progression of the disease. Therefore, further exploration of new therapeutic strategies is crucial not only for the clinical treatment of women with preeclampsia but also for the health management of their offspring.

[0003] Among the many pathogenic factors, endothelial cell damage is widely considered to be a core component of preeclampsia. Impaired placental vascular remodeling, excessive release of placental-derived factors, toxic substances, and inflammatory mediators lead to systemic endothelial cell damage, triggering systemic spasmodic contraction of small blood vessels, which in turn leads to a range of clinical symptoms such as hypertension, proteinuria, and edema. From local placental lesions to systemic multi-organ dysfunction, endothelial cells are not only directly damaged target cells but also key drivers of the development and progression of preeclampsia. Therefore, repairing and protecting endothelial cell damage has become an important research focus for the treatment of preeclampsia.

[0004] Endogenous peptides, a class of peptide molecules widely present in the body, are primarily derived from broken fragments of proteins. Although long considered intermediate products of protein processing or degradation, recent studies have revealed that endogenous peptides play important regulatory roles in immune regulation, cell proliferation and apoptosis, and neurotransmitter regulation. Mesenchymal stem cells are a type of multipotent stem cell with diverse differentiation potentials. Umbilical cord-derived mesenchymal stem cells possess multipotential differentiation potential, low immunogenicity, and low in vivo tumorigenicity, making them an important cell source for the study of alternative therapies and the isolation of endogenous peptides. However, no studies have yet identified endogenous peptides that can protect vascular endothelial cell function and alleviate the symptoms of preeclampsia. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an endogenous umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, nucleotide sequence and application, and the second purpose is to provide a drug and pharmaceutical composition containing the peptide.

[0006] Technical solution: The umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 of the present invention has the amino acid sequence shown in SEQ ID NO: 1.

[0007] Preferably, the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 is an endogenous polypeptide derived from dynein heavy chain 5

[0008] The nucleotide sequence of the present invention encodes the aforementioned umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2.

[0009] Use of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 of the present invention in the preparation of a drug for alleviating preeclampsia.

[0010] The invention provides an application of the umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 in the preparation of a drug for restoring and maintaining the integrity and function of vascular endothelium.

[0011] Preferably, the invention is used in the preparation of a drug for improving the angiogenesis ability of damaged endothelial cells.

[0012] Preferably, the invention is used in the preparation of a drug for reducing the expression of vascular endothelial injury markers.

[0013] The medicine of the present invention comprises the aforementioned umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 and a pharmaceutically acceptable salt thereof.

[0014] The pharmaceutical composition of the present invention comprises the aforementioned umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof.

[0015] Preferably, the aforementioned pharmaceutically acceptable carrier includes one or more of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an effervescent agent, a surfactant, an absorption enhancer, a lubricant, an adsorption carrier, a sustained-release microsphere, an implant, an in situ microsphere, a liposome, a microemulsion, an in situ hydrogel, and a nanoparticle.

[0016] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: 1. Umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 is an endogenous polypeptide with minimal toxic side effects; 2. It can effectively restore and maintain the integrity and function of the vascular endothelium, such as reducing the expression of vascular endothelial injury markers and improving the angiogenesis capacity of damaged endothelial cells; 3. It can effectively alleviate preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is the result of the activity of human umbilical vein endothelial cells treated with umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2;

[0018] Figure 2 The results of umbilical cord mesenchymal stem cell-derived anti-preeclampsia peptide 2 intervention to improve the angiogenesis of human umbilical vein endothelial cells are shown in Figure 1. A is a microscopic image, and B, from left to right, shows the statistical results of the number of vascular grid intersections, the number of vascular nodes, and the number of vascular grids.

[0019] Figure 3 This is the electron microscopic result of damaged human umbilical vein endothelial cells treated with umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2;

[0020] Figure 4 This is a graph showing the effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the gene expression of endothelial cell injury markers;

[0021] Figure 5 This is a graph showing the effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the expression of endothelial cell injury marker proteins;

[0022] Figure 6 The results of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 alleviating preeclampsia-like conditions in pregnant mice are shown in Figure 1, where A is a schematic diagram of the treatment process, B is a graph showing the blood pressure results of pregnant mice at different time points, C is a graph showing the urine protein content of pregnant mice at different time points, D is a comparison of fetal mice and placenta, and E is a graph showing the fetal weight results.

[0023] Figure 7 This is an analysis of the reasons why umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 alleviates preeclampsia-like symptoms in pregnant mice. A is the expression of vascular endothelial injury markers in the serum of pregnant mice, B is the H&E staining result of the pregnant mouse kidneys, and C is the H&E staining result of the pregnant mouse placenta. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below.

[0025] Example 1: Synthesis of Umbilical Cord Mesenchymal Stem Cell Anti-Preeclampsia Peptide 2 (APEP2)

[0026] Shanghai Kepeptide Biotechnology Co., Ltd. was commissioned to synthesize APEP2 (amino acid sequence as shown in SEQ ID NO: 1: AQTKRLVGDVLLATAFLSYSGP) by solid phase method.

[0027] The synthesized anti-preeclampsia peptide 2 was prepared into a 1 mM solution with DMSO and stored at -40°C for future use.

[0028] Example 2: Effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the activity of human umbilical vein endothelial cells

[0029] Cells were seeded at a density of 2000 cells per well in a 96-well plate. After the cells adhered, the culture medium was discarded. Complete culture medium containing APEP2 was added to a final concentration of 10 μM and the culture was continued. After culturing for 24 hours, the cells were removed, the culture medium was discarded, and complete culture medium containing 10% CCK-8 reagent was added. The cells were incubated in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. The results are shown in Figure 2. Figure 1 As shown in the results, APEP2 intervention had no significant effect on the viability of human umbilical vein endothelial cells.

[0030] Example 3: Effect of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on angiogenesis in damaged human umbilical vein endothelial cells

[0031] After adding matrix gel to the 96-well plate, it was placed in an incubator and allowed to stand for 45 minutes. After the gel solidified, 10,000 human umbilical vein endothelial cells were inoculated into each well and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium of the other groups contained TNF-α with a final concentration of 30 ng / ml. The TNF-α+Scr group was supplemented with a final concentration of 10 μM of scrambled peptide (Scr), and the TNF-α+APEP2 treatment group was supplemented with a final concentration of 10 μM of APEP2. Images were collected at regular intervals, and the tube length, coverage area, number of rings, and number of nodes were measured and recorded using ImageJ software. The results are shown in the figure below. Figure 2 As shown, APEP2 intervention can significantly improve the angiogenesis ability of damaged endothelial cells.

[0032] Example 4: Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on the morphology of damaged human umbilical vein endothelial cells

[0033] 10,000 human umbilical vein endothelial cells were seeded in each well of a 6-well plate and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium in the other groups contained TNF-α with a final concentration of 30 ng / ml. The TNF-α+Scr group was supplemented with a scrambled peptide (Scr) with a final concentration of 10 μM, and the TNF-α+APEP2 treatment group was supplemented with APEP2 with a final concentration of 10 μM. After culturing for 24 hours, the cells were digested, centrifuged, and the cell pellets were collected. Electron microscopy fixative was added, resuspended, mixed, and fixed at 4°C for 24 hours, and then handed over to Wuhan Savier Company for transmission electron microscopy. The results are shown in the figure. Figure 3As shown in the data, in the TNF-α injury group and the TNF-α+Scr group, the degree of cell edema was relatively obvious, manifested as matrix edema, mitochondrial membrane damage, matrix dissolution, and reduced rough endoplasmic reticulum. After APEP2 treatment, the degree of cell edema was relatively alleviated, the cytoplasm was abundant, the mitochondrial membrane structure was relatively intact and relatively abundant, and the rough endoplasmic reticulum did not expand, indicating that APEP2 can significantly improve the morphology of damaged endothelial cells.

[0034] Example 5: Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on endothelial cell injury markers

[0035] 1. Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on endothelial cell injury marker gene levels

[0036] 10,000 human umbilical vein endothelial cells were seeded in each well of a 6-well plate and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium of the other groups contained TNF-α with a final concentration of 30 ng / ml. The TNF-α+Scr group was supplemented with a final concentration of 10 μM scrambled peptide (Scr), and the TNF-α+APEP2 treatment group was supplemented with a final concentration of 10 μM APEP2.

[0037] After 24 hours, the 6-well plate was removed and washed with PBS. Total RNA was extracted using an RNA extraction kit and reverse transcribed. RT-qPCR was performed using a ChamQ Universal SYBR qPCR Master mix kit and a real-time fluorescence quantitative PCR system. GAPDH was used as an internal reference to calculate the relative expression of each gene in the cell. Two replicate wells were set up for each experiment and repeated three times. The results are shown in Figure 2. Figure 4 As shown in the figure, it can be seen that APEP2 can significantly inhibit the expression of vascular endothelial injury markers VCAM-1, ET-1, and sFlt-1 genes.

[0038] 2. Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on endothelial cell injury marker protein levels

[0039] 10,000 human umbilical vein endothelial cells were seeded in each well of a 6-well plate and divided into NC group, TNF-α injury group, TNF-α+Scr group, and TNF-α+APEP2 treatment group. Except for the NC group, the culture medium of the other groups contained TNF-α with a final concentration of 30 ng / ml. The TNF-α+Scr group was supplemented with a final concentration of 10 μM scrambled peptide (Scr), and the TNF-α+APEP2 treatment group was supplemented with a final concentration of 10 μM APEP2.

[0040] After 24 hours, the 6-well plate was removed and washed once with PBS. Protein was extracted by adding 100 μL / well of RIPA lysis buffer supplemented with 1% protease inhibitors and phosphatase inhibitors. After electrophoresis, the protein samples were transferred to a PVDF membrane. After blocking with blocking buffer, the membranes were incubated with mouse ET-1 and rabbit VCAM-1 primary antibodies, respectively, at 4°C for 16 hours. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies of the corresponding resistance at room temperature for 1 hour. After washing, enhanced chemiluminescence solution was added for development, and the resulting images were analyzed in grayscale.

[0041] The results are as follows Figure 5 As shown in the results, APEP2 can significantly inhibit the expression of vascular endothelial injury markers VCAM-1 and ET-1 proteins in human umbilical vein endothelial cells.

[0042] Example 6: Effects of umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 on preeclampsia-like mice

[0043] 1. Scr and APEP2 were modified by acetate conversion and endotoxin removal;

[0044] 2. Forty-nine healthy pregnant BALB / c mice housed under specific pathogen-free conditions were randomly divided into four groups: NC group, LPS model group, LPS+Scr group, and LPS+APEP2 treatment group;

[0045] 3. Systolic blood pressure was measured and urine was collected from each mouse on gestational days 5.5, 12.5, and 18.5. Pregnant mice in all groups, except the NC group, were intraperitoneally injected with 10 ng / g lipopolysaccharide (LPS) starting from gestational day 10.5 to establish a preeclampsia mouse model. Pregnant mice in the LPS+Scr group were also intraperitoneally injected with 10 mg / kg of scrambled polypeptide (Scr). Pregnant mice in the LPS+APEP2 group were also intraperitoneally injected with 10 mg / kg of APEP2.

[0046] 4. On day 18.5, all mice were euthanized with CO2, and ocular blood was collected for subsequent detection of vascular endothelial injury markers in the maternal circulation. The fetal placenta was weighed and photographed.

[0047] The results are as follows Figure 6 、 7 As shown, APEP2 significantly alleviated the preeclampsia-like phenotype in mice, lowered the blood pressure of the mothers, and alleviated the growth restriction of the offspring; H&E staining of the mouse kidneys showed that the glomerular capillaries in the LPS model group were occluded and the endothelial cells proliferated, while APEP2 treatment could improve kidney and placental damage and reduce the concentrations of vascular endothelial injury markers ET-1, sFlt-1 and TNF-α in maternal serum.

Claims

1. Use of an umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 having the amino acid sequence shown in SEQ ID NO: 1 in the preparation of a drug for alleviating preeclampsia.

2. Use of a pharmaceutical composition in the preparation of a drug for alleviating preeclampsia, characterized in that: The pharmaceutical composition comprises an umbilical cord mesenchymal stem cell anti-preeclampsia peptide 2 having an amino acid sequence as shown in SEQ ID NO: 1, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof.

3. The application according to claim 2, characterized in that: The pharmaceutically acceptable carrier includes one or more of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an effervescent agent, a surfactant, an absorption promoter, a lubricant, an adsorption carrier, a sustained-release microsphere, an implant, an in situ microsphere, a liposome, a microemulsion, an in situ hydrogel, and a nanoparticle.

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